The present invention relates to a missense-mutated protein at conserved amino acids in the PRPS1 (phosphoribosyl pyrophosphate synthetase 1) gene on Xq22.3 in two families with a syndromic form of inherited peripheral neuropathy, one of Asian and one of European descent, and to a mutation of the gene.
Charcot-Marie-Tooth (CMT) inherited neuropathy (CMT) represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. It is one of the most common inherited disorders of humans and is the most common genetic cause of neuropathy, with prevalence of 1 in 2500 people [see Skre H (1974) Genetic and clinical aspects of Charcot-Marie-Tooth's disease. Clin Genet 6:98-118]. Also, about 10-20% of CMT is inherited in an X-linked manner (CMTX). Male patients with this disease gene invariably develop sensorineural hearing loss of prelingual type followed by gating disturbance and visual loss. The family of European descent was reported in 1967 as having Rosenberg-Chutorian syndrome, and recently a Korean family with the same symptom triad was identified with a novel disease locus CMTX5 on the chromosome band Xq21.32-q24. Meanwhile, it was reported that patients suffering from RCS did not show mental retardation, like the case of CMTX5 [see Rosenberg R N, Chutorian A (1967) Familial opticoacoustic nerve degeneration and polyneuropathy. Neurology 17:827-832].
Among the five known loci for CMTX, the causal gene has been identified only in CMTX1 (MIM 302800), in which the neuropathy is caused by mutations in the gap junction protein beta 1 gene (GJB1 [MIM 304040) encoding the protein connexin 32 (Cx32) [see Bergoffen J, Scherer S S, Wang S, Scott M O, Bone L J, Paul D L, Chen K, Lensch M W, Chance P F, Fischbeck K H (1993) Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science 262:2039-2042]. A syndromic form of X-linked recessive CMT was identified in a Korean family, and the disease gene was localized to a novel locus for CMTX on the chromosome band Xq21.32-24 (CMTX5 [MIM 311070) [see Kim H J, Hong S H, Ki C S, Kim B J, Shim J S, Cho S H, Park J H, Kim J W (2005) A novel locus for X-linked recessive CMT with deafness and optic neuropathy maps to Xq21.32-q24. Neurology 64:1964-1967].
Meanwhile, in the present invention, the male patients with CMTX5 invariably developed a unique symptom triad of hearing loss, visual impairment, and peripheral neuropathy (see Table 1 and FIG. 1). The hearing loss is characterized as sensorineural and prelingual in nature. The patients experienced progressive visual impairment from optic neuropathy at about 10 years of age. At approximately the same age, the patients also experienced peripheral neuropathy that has been classified by electrophysiology as having mixed features of segmental demyelination and axonal loss. Follow-up of the youngest male patient (CMTX5:IV-11 in FIG. 1), who had been reported to have normal visual function at the time of initial presentation (at age 4 years), showed that his visual function began to be impaired at age 7 years. Pathologic examination of the sural nerve biopsy sample from the eldest living patient (CMTX5:III-18 in FIG. 1) showed loss of both large and small myelinated nerve fibers and an increase of endoneurial collagen (FIG. 3). Electron microscopic examination revealed segmental demyelination and remyelination with onion-bulb formation (FIG. 4). The three symptoms observed in CMTX5 were identical with that reported in Rosenberg-Chutorian syndrome (RCS) (Table 1 and FIG. 2), and it has been speculated that CMTX5 and RCS are allelic disorders sharing the common disease gene.
TABLE 1Characteristics of patients with CMTX5and RCS, a disease allelic to CMTX5AgeHearing lossVisual impairmentMotorDistalMNCV of medianUric acid levelbPatient(y)a/sex(Age at onset, y)(Age at onset, y)(Age at onset, y)sensory lossnerve (m s−1)(agec, y)CMTX5:III-1828/male+(Since infant)+(11)+(12)+NT4.48 (29)CMTX5:III-2125/male+(Since infant)+(13)+(12)−NTNTCMTX5:III-2418/male+(Since infant) +(8)+(12)−NTNTCMTX5:IV-114/male+(Since infant)+(11)+(10)−51.03.36 (14)CMTX5:IV-11 4/male+(Since infant)  +(7)d +(4)−43.14.73 (5) RCS:III-1132/male+(Since infant)+(20) +(5)+44.85.43 (70)RCS:III-1329/male+(Since infant)+(19) +(2)+46.53.53 (68)RCS:IV-143.5/male +(Since infant)−Nonspecific−NTNTNote:RCS = Rosenberg-Chutorian syndrome;MNCV = motor nerve conduction velocity;NT = Not tested.aAge at initial clinical evaluation and electrophysiological studies.b+ = positive finding; − = negative finding.cReference range, 3.40-7.20 mg/dL.dAge at the time of blood drawing for the determination of uric acid levels.eFollow-up examination of this patient (IV-11) showed decreased visual acuity 3 years after the initial evaluation (at age 7 years).
The present inventors have found that the disease locus of CMTX5 spans 15.2 cM and harbors more than 170 known genes and predicted genes. Accordingly, the present inventors have investigated the candidate genes that were known to be expressed in the inner ear according to the cochlear expression database (Morton cochlear expression database) within the locus, since sensorineural hearing loss has been the earliest symptom and sign of both CMTX5 and RCS [see Resendes B L, Robertson N G, Szustakowski J D, Resendes R J, Weng Z, Morton C C (2002) Gene discovery in the auditory system: characterization of additional cochlear-expressed sequences. J Assoc Res Otolaryngol 3:45-53].
Mutations identified in the present invention were E43D, in patients with Rosenberg-Chutorian syndrome, and M115T, in the Korean patients with CMTX5 (see SEQ ID NOs: 16 and 18). Also, enzyme activity decreased in patients with M115T.
Meanwhile, previous reports of missense mutations in PRPS1 have demonstrated increased rather than decreased enzyme activity, with “superactive” enzymes resulting in hyperuricemia and gout (MIM 311850) [see Becker M A, Smith P R, Taylor W, Mustafi R, Switzer R L (1995) The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J Clin Invest 96:2133-2141; and Becker M A, Taylor W, Smith P R, Ahmed M (1996) Overexpression of the normal phosphoribosylpyrophosphate synthetase 1 isoform underlies catalytic superactivity of human phosphoribosylpyrophosphate synthetase. J Biol Chem 271:19894-19899]. Also, it was reported that such mutations also caused sensorineural hearing loss and developmental delay [see Simmonds H A, Webster D R, Lingam S, Wilson J (1985), An inborn error of purine metabolism, deafness and neurodevelopmental abnormality. Neuropediatrics 16:106-108].
However, in the present invention, all serum uric acid levels in patients with CMTX5 and RCS were shown to have normal values (Table 1). No patient with CMTX5 or RCS had been diagnosed with gout or had other hyperuricemia-related symptoms or signs. These results demonstrate that the mutated PRPS1 of the present invention shows a decreased enzymatic activity, but can damage the nervous system without elevating uric acid levels. Accordingly, the use of a screening system with the mutated PRPS1 protein of the present invention allows the screening of drug candidates which increase or decrease the activity of PRPS1. The development of this screening system can be performed using a prior method for screening protein drugs.
Also, a kit, containing the mutated PRPS1 protein and a mutated gene thereof, can be used to diagnose acquired peripheral neuropathy. The kit can be prepared using any method known in the art.
The functional form of the PRPS1 enzyme was shown to have a hexameric structure. The PRPS1 monomer has five-stranded parallel β sheets and four α-helices on each of the N- and C-terminal domains, flanked by a short antiparallel β sheet protruding from the central core (a “flag” region) [see Eriksen T A, Kadziola A, Bentsen A K, Harlow K W, Larsen S (2000) Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 7:303-308].
In addition to catalytic and regulatory binding sites, PRPS1 has functional residues involved in intersubunit interactions and maintaining the stability of the enzyme. The mutation in CMTX5 occurred at the Met 115 residue of α-helix of N-terminal domain, and the mutation in RCS occurred at the Glu 43 residue of the “flag” region of the N-terminal domain (FIG. 10) [see Eriksen T A, Kadziola A, Bentsen A K, Harlow K W, Larsen S (2000) Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 7:303-308]. It was speculated that the increased enzyme activity was caused by mutations that alter the transmission of allosteric effects on the active sites of PRPS1, resulting in decreased inhibition [see Becker M A, Smith P R, Taylor W, Mustafi R, Switzer R L (1995) The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J Clin Invest 96:2133-2141].
In the present invention, the PRPS1 mutations causing decreased enzyme activity (CMTX5 and RCS) were occurring in the interface between the A and D subunits, whereas those causing increased enzyme activity were found in the interface between the A and C subunits, indicating that the different regulatory behaviors of mutations result from altered interactions between subunits (FIG. 11) [see Eriksen T A, Kadziola A, Bentsen A K, Harlow K W, Larsen S (2000) Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 7:303-308].
Meanwhile, acquired peripheral neuropathy, which can result from antimetabolite medications, is one of the major side effects of cancer chemotherapy and of other medications, such as statins and nucleoside reverse transcriptase inhibitors utilized to treat patients with HIV (drug-/chemotherapy-induced peripheral neuropathy) [see Peltier A C, Russell J W (2006) Advances in understanding drug-induced neuropathies. Drug Saf 29:23-30 and Sul J K, Deangelis L M (2006) Neurologic complications of cancer chemotherapy. Semin Oncol 33:324-332]. Some of these medications also cause sensorineural hearing loss and optic neuropathy, as in the patients with CMTX5 and RCS. Several agents have been developed for potential use as chemoprotectants to reduce these side effects, but none have proven effective [see Hausheer F H, Schilsky R L, Bain S, Berghorn E J, Lieberman F (2006) Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy. Semin Oncol 33:15-49].
Accordingly, the PRPS1 gene of the present invention is a metabolic enzyme critical for nucleotide biosynthesis, contributes to the pathophysiology of acquired peripheral neuropathy, and can be used as a therapeutic target to prevent or treat medication-induced acquired peripheral neuropathy, sensorineural hearing loss, and/or optic neuropathy [see Hausheer F H, Schilsky R L, Bain S, Berghorn E J, Lieberman F (2006) Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy. Semin Oncol 33:15-49 and Brinkman R R, Dube M P, Rouleau G A, Orr A C, Samuels M E (2006) Human monogenic disorders—a source of novel drug targets. Nat Rev Genet 7:249-260].
As described above, the present invention relates to the fact that missense mutations in the PRPS1 gene cause a syndromic form of peripheral neuropathy associated with sensorineural hearing loss and optic neuropathy. Accordingly, the present invention relates to inherited peripheral neuropathy resulting from decreased activity of a metabolic enzyme critical for purine metabolism and nucleotide biosynthesis, and relates to the understanding of peripheral nerve-specific metabolism and the treatment of antimetabolite-induced acquired peripheral neuropathy.